1. Field of the Invention
This invention relates to methods and apparatus for performing microanalytic analyses and procedures on fluid samples. In particular, the invention relates to microminiaturization of genetic, biochemical and chemical processes related to analysis, synthesis and purification. Specifically, the invention provides a microsystem platform that is rotationally manipulated by a micromanipulation device, thereby utilizing the centripetal forces resulting from rotation of the platform to motivate fluid movement through microchannels embedded in the microplatform. The microsystem platforms of the invention are provided having microfluidics components, chambers and reservoirs, resistive heating elements, temperature sensing elements, mixing structures, capillary and sacrificial valves, and methods for using these microsystems platforms for performing biological, enzymatic, immunological and chemical assays.
2. Summary of the Related Art
Assays for detecting analytes in fluid samples, particularly complex fluids such as biological fluid samples, are used for a variety of diagnostic, environmental, synthetic and analytical purposes in the medical, biological, chemical, biochemical and environmental arts.
Certain analytes are important for diagnosis and monitoring of acute and chronic disease in humans. For example, diabetes is the fourth major cause of morbidity and mortality in the U.S., even though the biochemical basis for the disease has been known for at least a century and drugs (primarily insulin) and methods for managing the disease are robust and widely used. It has been recognized that sensitive monitoring of blood sugar levels, either directly or by detecting levels of metabolites and disease-associated modifications (such as the relative fraction of glycated hemoglobin in the bloodstream) is important in managing the disease. However, fast and inexpensive ways of performing the assays necessary for efficient management of multiple indicators of the disease state are not currently available.
In the field of medical, biological and chemical assays, mechanical and automated fluid handling systems and instruments are known in the prior art.
U.S. Pat. No. 4,279,862, issued Jul. 21, 1981 to Bertaudiere et al. disclose a centrifugal photometric analyzer.
U.S. Pat. No. 4,381,291, issued Apr. 26, 1983 to Ekins teach analytic measurement of free ligands.
U.S. Pat. No. 4,515,889, issued May 7, 1985 to Klose et al. teach automated mixing and incubating reagents to perform analytical determinations.
U.S. Pat. No. 4,676,952, issued Jun. 30, 1987 to Edelmann et al. teach a photometric analysis apparatus.
U.S. Pat. No. 4,745,072, issued May 17, 1998 to Ekins discloses immunoassay in biological fluids.
U.S. Pat. No. 5,061,381, issued Oct. 29, 1991 to Burd discloses a centrifugal rotor for performing blood analyses.
U.S. Pat. No. 5,122,284, issued Jun. 16, 1992 to Braynin et al. discloses a centrifugal rotor comprising a plurality of peripheral cuvettes.
U.S. Pat. No. 5,160,702, issued Nov. 3, 1993 to Kopf-Sill and Zuk discloses rotational frequency-dependent valves using capillary forces and siphons, dependent on xe2x80x9cwettabilityxe2x80x9d of liquids used to prime said siphon.
U.S. Pat. No. 5,171,695, issued Dec. 15, 1992 to Ekins discloses determination of analyte concentration using two labeling markers.
U.S. Pat. No. 5,173,193, issued Dec. 22, 1992 to Schembri discloses a centrifugal rotor for delivering a metered amount of a fluid to a receiving chamber on the rotor.
U.S. Pat. No. 5,242,803, issued Sep. 7, 1993 to Burtis et al. disclose a rotor assembly for carrying out an assay.
U.S. Pat. No. 5,409,665, issued Apr. 25, 1995 to Burd discloses a cuvette filling in a centrifuge rotor.
U.S. Pat. No. 5,413,009, issued Jul. 11, 1995 to Ekins discloses a method for analyzing analytes in a liquid.
U.S. Pat. No. 5,472,603, issued Dec. 5, 1995 to Schembri discloses an analytical rotor comprising a capillary passage having an exit duct wherein capillary forces prevent fluid flow at a given rotational speed and permit flow at a higher rotational speed.
Anderson, 1968, Anal. Biochem. 28: 545-562 teach a multiple cuvette rotor for cell fractionation.
Renoe et al., 1974 Clin. Chem. 20: 955-960 teach a xe2x80x9cminidiscxe2x80x9d module for a centrifugal analyzer.
Burtis et al., 1975, Clin. Chem. 20: 932-941 teach a method for a dynamic introduction of liquids into a centrifugal analyzer.
Fritsche et al., 1975, Clin. Biochem. 8: 240-246 teach enzymatic analysis of blood sugar levels using a centrifugal analyzer.
Burtis et al., 1975, Clin Chem. 21: 1225-1233 teach a multipurpose optical system for use with a centrifugal analyzer.
Hadjiioannou et al., 1976, Clin. Chem. 22: 802-805 teach automated enzymatic ethanol determination in biological fluids using a miniature centrifugal analyzer.
Lee et al., 1978, Clin. Chem. 24: 1361-1365 teach a automated blood fractionation system.
Cho et al., 1982, Clin. Chem. 28: 1956-1961 teach a multichannel electrochemical centrifugal analyzer.
Bertrand et al., 1982, Clinica Chimica Acta 119: 275-284 teach automated determination of serum 5xe2x80x2-nucleotidase using a centrifugal analyzer.
Schembri et al., 1992, Clin Chem. 38: 1665-1670 teach a portable whole blood analyzer.
Walters et al., 1995, Basic Medical Laboratory Technologies, 3rd ed., Delmar Publishers: Boston teach a variety of automated medical laboratory analytic techniques.
Recently, microanalytical devices for performing select reaction pathways have been developed.
U.S. Pat. No. 5,006,749, issued Apr. 9, 1991 to White disclose methods apparatus for using ultrasonic energy to move microminiature elements.
U.S. Pat. No. 5,252,294, issued Oct. 12, 1993 to Kroy et al. teach a micromechanical structure for performing certain chemical microanalyses.
U.S. Pat. No. 5,304,487, issued Apr. 19, 1994 to Wilding et al. teach fluid handling on microscale analytical devices.
U.S. Pat. No. 5,368,704, issued Nov. 29, 1994 to Madou et al. teach microelectrochemical valves.
International Application, Publication No. WO93/22053, published Nov. 11, 1993 to University of Pennsylvania disclose microfabricated detection structures.
International Application, Publication No. WO93/22058, published Nov. 11, 1993 to University of Pennsylvania disclose microfabricated structures for performing polynucleotide amplification.
Columbus et al., 1987, Clin. Chem. 33: 1531-1537 teach fluid management of biological fluids.
Ekins et al., 1994 Ann. Biol. Clin. 50: 337-353 teach a multianalytic microspot immunoassay.
Wilding et al., 1994, Clin. Chem. 40: 43-47 disclose manipulation of fluids on straight channels micromachined into silicon.
One drawback in the prior art microanalytical methods and apparati has been the difficulty in designing systems for moving fluids on microchips through channels and reservoirs having diameters in the 10-500 xcexcm range. Microfluidic systems require precise and accurate control of fluid flow and valving to control chemical reactions and analyte detection. Conventional pumping and valving mechanisms have been difficult to incorporate into microscale structures due to inherent conflicts-of-scale. These conflicts of scale arise in part due to the fact that molecular interactions arising out of mechanical components of such components, which are negligible in large (macroscopic) scale devices, become very significant for devices built on a microscopic scale.
While devices and pumping and valving mechanisms have been developed which overcome some of these conflict-of-scale difficulties, there are other inherent problems with these systems. A number of microanalytical platforms have been developed which use electrokinetic forces for fluid pumping: electroosmotic flow devices; electrohydrodynamic devices; and electrophoretic devices. An inherent drawback in these systems is that they rely on precise control of pH and free charges in the fluid being pumped. This makes them incapable of pumping most raw biological samples, such as blood and urine, and creates difficulties in pumping organic solvents. In cases where these systems may be used, pre-conditioning of the fluid to enhance electrokinetic effects is usually required.
Systems that use centripetal force to effect fluid movement in microstructures address the need for a pumping mechanism to effect fluid flow, but cannot alone solve these scale-related drawbacks of conventional fluidics reduced to microfluidics scale. There remains a need for a simple, flexible, reliable, rapid and economical microanalytic and microsynthetic reaction platform for performing biological, biochemical and chemical analyses and syntheses that can move fluids within the structural components of a microsystems platform. Such a platform should be able to move nanoliter-to microliter amounts of fluid, including reagents and reactants, at rapid rates to effect the proper mixing of reaction components, removal of reaction side products, and isolation of desired reaction products and intermediates. There remains a need in the art for centripetally-motivated microfluidics platforms capable of precise and accurate control of flow and metering of fluids in both microchip-based and centrifugal microplatform-based technologies.
This invention provides Microsystems platforms as disclosed in co-owned and co-pending U.S. Ser. No. 08/761,063, filed Dec. 5, 1996 and U.S. Ser. No. 09/083,678, filed May 22, 1998, each of which is incorporated by reference herein. Specifically, this invention provides microfluidics platforms for performing biological, enzymatic, immunological and chemical assays.
It is an advantage of the centrifugal rotors and Microsystems platforms of the invention that an imprecise amount of a fluid comprising a biological sample can be applied to the rotor or platform and a precise volumetric amount of the biological sample is delivered to a fluid reservoir comprising a reaction vessel or other component of the rotor or platform for performing chemical, biochemical, immunological or other analyses. It is an advantage of the centrifugal rotors and microsystems platforms of the invention that metering of said precise amount of a biological fluid sample, for example, a drop of blood, is provided as an intrinsic property of the metering capillary channel of the rotor or platform, thereby avoiding variability introduced by centripetal metering of the sample into a reaction reservoir. It is a further advantage of the centrifugal rotors and Microsystems platforms of the invention that an operator can avoid having to precisely measure an amount of a fluid comprising a biological sample for application to the rotor or microsystem platform, thereby permitting end-users, including consumers, having a lower level of sophistication to use a medically diagnostic or other embodiment of the rotor or microsystem platform of the invention.
It is an advantage of the centrifugal rotors and Microsystems platforms of the invention that fluid movement into and out of fluid reservoirs on the rotor or platform is precisely determined by displacement of a first fluid, such as biological sample, from a fluid reservoir by a second fluid contained in a second reservoir on the rotor or platform. It is also an advantage of the centrifugal rotors and Microsystems platforms of the invention that approximately complete replacement of the volumetric capacity of a first reservoir can be achieved by using fluid displacement as disclosed herein, thereby providing for maximum recovery of a first fluid sample upon displacement by a second fluid, or maximum delivery and replacement of the first fluid by the second fluid. This aspect of the invention is advantageous for providing sequential chemical or biochemical reaction steps wherein mixing of the reagents is not desired.
It is also an advantage of the centrifugal rotors and Microsystems platforms of the invention that these platforms provide an integrated microfluidics system containing components and structures for performing microanalytic assays whereby fluid flow on the platform is motivated by centripetal force and controlled by capillary and/or sacrificial valves. The invention provides such integrated platforms whereby an operator is required simply to apply a sample, most preferably an imprecise volume of a fluid sample, to an entry port on the disk surface, and a complex series of analytical steps are performed without further operator manipulation on the platform. Movement of fluids on the disk, and the sequence of analytical reaction steps performed thereupon, is a consequence of changes in rotor speed and/or the opening of sacrificial valves as directed by an instruction set contained in a program contained on the disk itself or in the memory of the micromanipulation apparatus that controls disk rotation and performance.
The Microsystems platforms also provide disks that can perform a multiplicity of analytical reactions on either several samples or a particular sample, whereby the reactions are performed sequentially or individually. In addition, a wide variety of analytic reactions can be performed on the microsystems platforms of the invention, as further described below.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.